Enterohemorrhagic E. coli (EHEC) O157:H7 is recognized as an important emerging pathogen responsible for producing hemorrhagic colitis and the hemolytic uremic syndrome (HUS) in humans. EHEC O157:H7 strains elaborate a potent Shiga toxin, which has been associated with the pathogenesis of HUS. No pili have yet been reproducibly identified in O157:H7 strains and therefore, it is still an enigma as to whether pili play a role in colonization of the intestine of their natural bovine or accidental human hosts. We have recently identified a novel pilus produced by EHEC strain EDL933 and other O157:H7 strains. These pili, herein called hemorrhagic coli pili 1 (Hcp1), belong to the virulence-associated type IV pili family. Further, purified Hcp1 showed agglutination of rabbit erythrocytes and bound to fibronectin and laminin suggesting an adhesive role in binding to human proteins. An isogenic Hcp1-deficient mutant showed considerably low levels of adherence to cultured cells. The pilin monomer is encoded by the chromosomal prepilin peptidase-dependent ppdD gene, which is adjacent to the type IV-like piliation genes hofBC. Based on our preliminary data, we hypothesize that Hcp1 is a key virulence factor that contributes to the adhesive properties of EHEC. Overall, the objective of this proposal is to advance knowledge of EHEC pathogenesis by elucidating the mechanism(s) of adherence of EHEC O157:H7 to human epithelial cells in culture. Several multidisciplinary approaches involving molecular biology, cell biology, ultrastructural analysis by high power electron microscopy, and biochemical and antigenic analysis will be carried out to extend our current knowledge on the interaction of EHEC O157:H7 with host target cells. The outcome of this proposal will provide important implications for detection of E. coli O157:H7 in food sources and reservoirs and importantly for prevention and control of EHEC infections in humans. The central focus of this proposal lies in structure-function studies of Hcp produced by EHEC. Thus, the following specific aims are proposed: 1) To define the genes required for Hcp1 biogenesis; 2) To define the role of Hcp1; 3) To analyze expression of ppdD.